It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and man. For disease states, classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect might be obtained with minimal side effects. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides, especially oligonucleotides which are complementary to a specific target messenger RNA (mRNA) sequence. Several oligonucleotides are currently undergoing clinical trials for such use.
Transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate the action of transcription factors. Several recent reports describe such interactions (see Bielinska, A., et. al., Science, 1990, 250, 997-1000; and Wu, H., et. al., Gene, 1990, 89, 203-209).
In addition to such use as both indirect and direct regulators of proteins, oligonucleotides have also found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with gene expression inhibition, diagnostic applications utilize the ability of oligonucleotides to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligonucleotides via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
Oligonucleotides are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other biological molecules. For example, the use of oligonucleotides as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits which assist non-molecular biology-trained personnel in applying PCR. Oligonucleotides, both natural and synthetic, are employed as primers in PCR technology.
Oligonucleotides are also used in other laboratory procedures. Several of these uses are described in common laboratory manuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel, et al., Eds., Current Publications, 1993. Such uses include Synthetic Oligonucleotide Probes, Screening Expression Libraries with Antibodies and Oligonucleotides, DNA Sequencing, In Vitro Amplification of DNA by the Polymerase Chain Reaction and Site-directed Mutagenesis of Cloned DNA (see Book 2 of Molecular Cloning, A Laboratory Manual, ibid.) and DNA-Protein Interactions and The Polymerase Chain Reaction (see Vol. 2 of Current Protocols In Molecular Biology, ibid).
Oligonucleotides can be synthesized to have custom properties that are tailored for a desired use. Thus a number of chemical modifications have been introduced into oligonucleotides to increase their usefulness in diagnostics, as research reagents and as therapeutic entities. Such modifications include those designed to increase binding to a target strand (i.e. increase their melting temperatures, (Tm)); to assist in identification of the oligonucleotide or an oligonucleotide-target complex; to increase cell penetration; to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides; to provide a mode of disruption (terminating event) once sequence-specifically bound to a target; and to improve the pharmacokinetic properties of the oligonucleotides.
Thus, it is of increasing value to prepare oligonucleotides and other phosphate linked oligomers for use in basic research or for diagnostic or therapeutic applications. Consequently, and in view of the considerable expense and time required for synthesis of specific oligonucleotides, there has been a longstanding effort to develop successful methodologies for the preparation of specific oligonucleotides with increased efficiency and product purity.
Synthesis of oligonucleotides can be accomplished using both solution phase and solid phase chemistries. Oligonucleotide synthesis via solution phase in turn can be accomplished with several coupling chemistries. However, solution phase chemistry requires purification after each internucleotide coupling, which is labor intensive and time consuming.
The current method of choice for the preparation of naturally occurring oligonucleotides, as well as modified oligonucleotides such as phosphorothioate and phosphorodithioate oligonucleotides, is via solid-phase synthesis wherein an oligonucleotide is prepared on a polymer support (a solid support) such as controlled pore glass (CPG); oxalyl-controlled pore glass ( see, e.g., Alul, et al., Nucleic Acids Research 1991, 19, 1527); TENTAGEL Support, (see, e.g., Wright, et al., Tetrahedron Letters 1993, 34, 3373); or POROS, a polystyrene resin available from Perceptive Biosystems.
Solid-phase synthesis relies on sequential addition of nucleotides to one end of a growing oligonucleotide chain. Typically, a first nucleoside (having protecting groups on any exocyclic amine functionalities present) is attached to an appropriate glass bead support and activated phosphite compounds (typically nucleotide phosphoramidites, also bearing appropriate protecting groups) are added stepwise to elongate the growing oligonucleotide. The nucleotide phosphoramidites are reacted with the growing oligonucleotide using the principles of a "fluidized bed" for mixing of the reagents. The known silica supports suitable for anchoring the oligonucleotide are very fragile and thus cannot be exposed to aggressive mixing. Brill, W. K. D., et al. J. Am. Chem. Soc., 1989, 111, 2321, disclosed a procedure wherein an aryl mercaptan is substituted for the nucleotide phosphoramidite to prepare phosphorodithioate oligonucleotides on glass supports.
Additional methodologies utilizing solid-phase synthesis may be found in Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Pat. Nos. 4,725,677 and Re. 34,069. In these and other solid-phase procedures the oligonucleotide is synthesized as an elongating strand. However, the number of individual strands that can be anchored to a unit surface area of the support is limited. Also, the activated nucleotides that are added to the growing oligonucleotide are relatively expensive and must be used in stoichiometric excess.
While presently-utilized solid-phase syntheses are very useful for preparing small quantities of oligonucleotide, i.e., from about the .mu.mol to mmol range, they typically are not amenable to the preparation of the larger quantities of oligonucleotides necessary for biophysical studies, pre-clinical and clinical trials and commercial production. Currently, to synthesize more than about three fourths of a mmol of oligonucleotide it is necessary to do sequential syntheses. A general review of solid-phase versus solution-phase oligonucleotide synthesis is given in the background section of Urdea, et al. U.S. Pat. No. 4,517,338, entitled "Multiple Reactor System And Method For Oligonucleotide Synthesis"
Solution-phase synthetic oligonucleotide techniques should be useful for large scale preparation. One such solution phase preparation utilizes phosphorus triesters. As reported in Yau, E. K., et.al., Tetrahedron Letters, 1990, 31, 1953, the triester oligonucleotide approach was used to prepare thymidine dinucleoside and thymidine dinucleotide phosphorodithioates. The phosphorylated thymidine nucleoside intermediates utilized in this approach were obtained by treatment of commercially available 5'-O-dimethoxytritylthymidine-3'- (.beta.-cyanoethyl)-N,N-diisopropyl!-pho sphoramidite first with either 4-chloro or 2,4-dichlorobenzylmercaptan and tetrazole, and then a saturated sulfur solution. The resulting phosphorodithioate nucleotide was then reacted via the triester synthesis method with a further thymidine nucleoside having a free 5'-hydroxyl.
Brill, W. K. D., et.al., J. Am. Chem. Soc., 1991, 113, 3972, recently disclosed that treatment of a phosphoramidite such as N,N-diisopropyl phosphoramidite with a mercaptan such as 4-chloro or 2,4-dichlorobenzylmercaptan in the presence of tetrazole yields a derivative suitable for preparation of a phosphorodithioate as a major product and a derivative suitable for preparation of a phosphorothioate as a minor product.
Further details of methods useful for preparing oligonucleotides may be found in Sekine, M., etc al., J. Org. Chem., 1979, 44, 2325; Dahl, O., Sulfur Reports, 1991, 11, 167-192; Kresse, J., et.al., Nucleic Acids Research, 1975, 2, 1-9; Eckstein, F., Ann. Rev. Biochem., 1985, 54, 367-402; and Yau, E. K. U.S. Pat. No. 5,210,264 entitled "S-(2,4-Dichlorobenzyl)-.beta.-Cyanoethyl Phosphorothioate Diester".
Although phosphorothioates and other oligonucleotides are of great utility, the art suggests no large scale techniques for their preparation. Accordingly, there remains a long-felt need for such methods, and particularly for methods having improved efficiency.
The yield improvements obtained with the present invention are achieved specifically with regard to the deblocking procedures for the 5' hydroxyl group and in less frequently the 3' hydroxyl group of the oligonucleotide being synthesized, and more particularly with regard to the use of a carbocation scavenging agent for any one or more of the protecting groups conventionally used for blocking the 5' hydroxyl and 3' hydroxyl groups. The use of such a scavenging agent has not been suggested heretofore in the art concerning synthesis of oligonucleotides, such as is represented by the technical literature and U.S. patents set out further above. The level of yield improvement which has been achieved with the use of such scavenging agents is all the more surprising in view of the longstanding conventional use of protecting groups such as dimethoxytrityl and other similar groups.
Although scavenging groups have been used with trityl blocking groups in syntheses which are in no way analogous to the oligonucleotide syntheses described above, it was an unexpected discovery that it was possible to achieve significant increases in yield by using such scavenging agents with trityl blocking groups in the synthesis of oligonucleotides. Representative of such non-analogous art is the following:
Mehta, A., et al., Tetrahedron Letters, 1992, 33, 5441-5444, discloses improved selectivity of deprotection of Boc and related t -butyl -containing protecting groups in peptide synthesis, where deprotection is by trifluoroacetic acidmediated acidolysis, through the use of triethylsilane and other carbocation scavengers;
Kemp, D. S., et al., J. Peptide Protein Res., 1998, 31, 359-372, discloses a study of the deblocking of N-a-Bpoc peptides in dichloromethane containing 0.5% trifluoroacetic acid, which found that only indole, benzenethiol, and benzyl mercaptan showed moderate reactivity as carbocation scavengers, while phenol, resorcinol, 1,3 -dimethoxybenzene, 1,3,5 -trimethoxybenzene, and dimethyl sulfide were inefficient.
Pearson, D. A., et al., Tetrahedron Letters, 1989, 30, 2739-2742, discloses the use of triethylsilane and triisopropylsilane as carbocation scavengers in the acidic deblocking of trityl blocked sulfhydryl groups in peptide synthesis.
Brenner, D., et al., Inorg. Chem., 1984, 23, 3793-3797, and U.S. Pat. No. 4,673,562, discloses the use of triethylsilane during trifluoroacetic acid deblocking of a trityl protected mercaptoacetic acid derivative in a step for preparing oxotechnetium (V) diamido dithioates.